Abstract Buffalo milk has been increasingly explored in the production of dairy foods. Given the diversity of lactic acid bacteria present in this raw material, the functional potential of this type of milk should be studied. This study aims to assess the probiotic potential of two strains of Enterococcus faecium isolated from buffalo milk. We evaluated E. faecium M7AN7-1 and E. faecium M7AN10 for sustained cell viability under different conditions of conservation. We also assessed adhesion and cell viability in a simulated gastrointestinal transit test. E. faecium M7AN10 was selected for microencapsulation in sodium alginate and testing in the food matrix. The isolates maintained cell viability after refrigeration, freezing, and freeze-drying. E. faecium M7AN10 showed higher concentrations of viable cells than E. faecium M7AN7-1 after 180 min of contact with simulated gastric fluid, reaching 7.19 ± 0.59 Log10 CFU mL-1. We also observed sustained cell viability after exposure to simulated intestinal fluid. After encapsulation, E. faecium M7AN10 was tested in the food matrix of UHT milk. Cell count viability was maintained and its release was sustained in this medium for 30 days. The results of the in vitro assessment demonstrated that E. faecium M7AN10 remained functionally active under these experimental conditions. Similarly, they showed that it is a bacterium capable of sustaining viability in this type of food and after transit in a simulated gastrointestinal tract. Based on the data, we suggest this isolate may be a probiotic bacterial candidate for in vivo behavioral assessment.
Keywords: lactic acid bacteria; Enterococcus; buffalo milk; microencapsulation; probiotics
*Address for Correspondence: Amanda de Souza da Motta, Microbiology Laboratory, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil, Tel: +55 51 33084111; E-mail: amanda.motta@ufrgs.br; ORCID ID: https://orcid.org/0000-0001-8637-3868 Received: 16 December 2020; Accepted: 12 March 2021; Published: 27 March 2021 Citation of this article: Kurtz, JP., Paim, WP., Jantzen, MM., Motta., AS. (2021) The Probiotic Properties of Isolated From Buffalo Milk: Food Matrix Studies. J Clin Nutr Food Sci, 4(1): 17-29.
Copyright: © 2021 Amanda de Souza da Motta, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
-1 Log CFU mL . We also observed sustained cell viability in Based on the data, we suggest this isolate may be a probiotic bacterial candidate for gastrointestinal tract.
assessment.
Keywords: lactic acid bacteria; Enterococcus; buffalo milk; microencapsulation;
Buffalo milk possesses a food matrix with high levels of protein, lipid, lactose and some minerals. It also has a higher nutritional content than bovine milk, particularly protein and total solids levels. Gradually, buffalo milk has been gaining ground in the development of new food products. However, little is known about the lactic acid microbiota and the functional properties of this raw material [1,2].
Evaluating the probiotic characteristics of lactic acid bacteria (LAB) involves multiple steps and takes time. One study evaluated the properties of four lactic acid bacteria that were candidates for probiotics. Lactobacillus casei SJRP35, Leuconostoc citreum SJRP44, Lactobacillus delbrueckii subsp. bulgaricus SJRP57 and Leuconostoc mesenteroides subsp. mesenteroides SJRP58. The strains survived at different pH values and bile salt concentration, and present autoaggregation and co-aggregation properties. The strain SJRP57 was considered the best candidate as probiotic cultures for further in vivo studies because did not show the presence of any genes encoding virulence factors [3]. Lactobacillus rhamnosus LB 1.5 and Lactobacillus paracasei LB 6.4 were buffalo milk isolates and researchers have recognized their probiotic potential and studied them in food tests [2].
The advancement of technologies seeking the better use and functional performance of lactic bacteria in products has given rise to the microencapsulation process [4]. The microencapsulation of probiotic bacteria has been described in the literature and has shown promising results. A research encapsulated ST16Pa in sodium alginate at concentrations of 2, 3, and 4%, using the extrusion method. They evaluated the exposure of free and encapsulated cultures under simulated gastric conditions. The authors found that alginate protected the bacterial culture, regardless of the concentration used. Moreover, they measured the decrease in the viability of encapsulated microorganisms at 1.2 logs, while the free culture had its count reduced by 5 logs in vivo behavioral probiotics [5]. Similarly, were encapsulated L. paracasei L26, L. casei-01, L.
-1 acidophilus Ki and B. animalis BB 2 in 2% alginate and stored the microcapsules at temperatures of 21, 4, -20 and -80 ºC for six months. The results demonstrated that the loss of viability was lower in microcapsules stored at freezing temperatures [6]. According to these studies, the use of probiotics in dairy products is an important alternative for the promotion of new functional foods. Research concerning indigenous microorganisms is relevant since milk contains microbiota with many potential applications.
Previous to this study, we had already isolated the LAB from buffalo milk and identified them as Enterococcus faecium M7AN7- 1 and Enterococcus faecium M7AN10. Their sequences were deposited in the Standard Nucleotide BLAST (https://blast.ncbi. nlm.nih. gov/) under the codes MH723756 and MH723757, respectively. These bacteria were tested at a freezing temperature of -20°C, in skim milk and 10% glycerol. The LAB were reactivated using an MRS (Man, Rogosa and Sharpe) broth culture and incubated at 35ºC for 48 h. After, the cultures on the MRS agar medium were depleted and incubated at 35ºC for 48 h to evaluate their purity. A commercial, probiotic culture of Lactobacillus rhamnosus (Fagron®) was used as a control.
For refrigeration, a sterile solution of distilled water and 10% skimmed-milk powder was prepared. To test freezing and freeze-drying, a sterile solution of distilled water and 20% skimmed-milk powder was used. The bacteria were inoculated in microtubes in these respective solutions, starting with an -1 initial inoculum of 8.00 Log CFU mL . Microtubes were refrigerated for 30 days or frozen for 90 days, a period during which the cultures were thawed and a viable cell count was carried out monthly [7]. The microtubes containing lyophilized cultures were stored in a freezer for six months, and cell viability was evaluated monthly. After these treatments, testing for viable bacteria cells was performed using plates incubated at 35 ± 1ºC for 24 h. Tests were -1 done in duplicate and results were expressed in Log CFU mL [8].
Solutions containing MRS broth and bile salts were prepared in concentrations of 0.1%, 0.3%, 0.5%, and 1%. Afterward, 1000 μL of these solutions were transferred to microtubes, and 100 μL of bacterial inoculum was added. These were incubated at 35 ± 1ºC for 4 h. The control treatment used MRS broth with no added bile salts. Viable cells were counted at the beginning of the test after the 4 h period, on plates incubated at 35 ± 1ºC for 24 h. Tests were done in triplicate [9].
The test to assess the tolerance of lactic acid bacteria to simulated gastric fluid (SGF) was carried out, with some modifications [10]. This fluid was prepared using a 0.5 % NaCl solution and 0.3 g mL -1 of pepsin, with the pH adjusted to 2 with 0.1 N HCl. In microtubes, 300 μL of a 0.5% NaCl solution with 200 μL of bacterial inoculum were added. Then 500 μL of SGF were placed in these same wells. The control group was tested with 500 μL of a 0.5% NaCl solution instead of SGF. The microtubes were incubated at 35 ± 1ºC for 180 min. Aliquots of 100 μL were sampled at the beginning of the test and at 60, 90, and 180 min, to obtain viable cell counts at different times. The plates were incubated at 35 ± 1ºC for 24 h and the assay was performed in triplicate.
The simulated intestinal fluid (SIF) was a solution containing 0.5% -1 NaCl and 0.1 g mL of pancreatin. The pH was adjusted to 8 with 0.1 N NaOH. We added 300 μL of a 0.5% NaCl solution and 200 μL of the bacterial inoculum in microtubes. Afterward, 500 μL of SIF was added. The control group was tested with 500 μL of a 0.5% NaCl solution instead of SIF, and the microtubes were incubated at 35 ± 1ºC for 240 min. A viable cell count was performed at the beginning of the test, and after 60, 90, 180, and 240 min, using plates incubated at 35 ± 1ºC for 24 h. The experiment was realized in triplicate with some modifications [10].
We tested LAB adhesion to Vero cells (the renal cells of the African green monkey Cercopithecus aethiops) that were kept in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine -1 -1 serum, 10,000 U mL of penicillin, and 10,000 U ml streptomycin. They were incubated at 37°C and in an atmosphere of 5% CO . When the Vero cells reached confluence, they were added to a 96-well microplate for cell cultivation, at a concentration of 2 x 10 cells, and incubated under the same conditions mentioned above. Afterward, the MEM was removed from the wells and they were washed with 200 μL PBS buffer, pH 7.2. LAB cultures were inoculated in MRS broth for 24 h at 35 ± 1ºC. Afterwards, these cultures were centrifuged at 4000 rpm, at a temperature of 4ºC for 10 min. The supernatant was discarded and the precipitate was washed twice with PBS buffer and resuspended in antibioticfree MEM. 100 μL from each culture were inoculated in wells containing Vero cells and the microplate was incubated at 37ºC for 4 h. After this period, the microplate was taken out from the oven and the bacterial suspension was removed. The wells were washed three times with 200 μL PBS buffer to remove non-adherent bacterial cells. Then, adhesion was interrupted by adding 200 μL of 0.5% Triton X100. The bacterial cell count was performed by incubating the plates at 35 ± 1ºC for 24 h. The percentage of adhered bacterial cells to Vero cells was calculated based on the relationship between the number of adhered bacteria and the number added at the beginning of the test. This test was carried out in triplicate [11-13].
For this procedure, 5 mL of the bacterial inoculum were centrifuged at 2000 rpm, for 10 min at 4ºC. Afterward, the cells were washed twice with 0.1% sterile peptone water and resuspended in 5 mL of the same solution to standardize the initial inoculum. A solution with a volume of 25 mL was prepared with 2% sodium alginate in distilled water. It was added to the cells that were previously resuspended. This content was vortexed and transferred to a sterile syringe equipped with a 0.45 mm diameter needle. The liquid was ejected into a sterile solution with 100 ml of 0.05M CaCl supplemented with 0.1% Tween 80. The solution with the microcapsules was centrifuged at 350 rpm, at 4ºC for 10 min and washed with 0.1% sterile peptone water, and filtered through Grade 1 Whatman filter paper. The microcapsules were transferred to a flask containing 100 mL of 0.1% sterile peptone water to further test encapsulation efficiency. This test was carried out according to the methodology proposed [5].
We added 1 g of sodium alginate microcapsules to 9 mL of a 2% sodium citrate solution. This content was centrifuged at 300 rpm, at a temperature of 17ºC for 10 min to release the microbial culture. After that, serial dilution was performed to -1 determine CFU g . Encapsulation efficiency was expressed in percentages using the following formula: Efficiency = YZ / X x100, where X represents the initial microorganism count before encapsulation; Y the count of encapsulated microorganisms and Z the count of microorganisms in the external fluid around the capsules (i.e. in the 0.05M CaCl solution) [6]. This measure was calculated according authors [14].
This experiment was carried out in test tubes with 1 g of microcapsules and 9 mL of separate samples of whole UHT cow milk, MRS broth, and a commercial, chocolate-flavored UHT milk drink. Test tubes were kept in refrigerated storage (7°C). The release of the LAB microbial cultures was evaluated when the microcapsules were added to these liquids and after 7, 15, 20, and 30 days of the test. Each tube was vortexed for five seconds for homogenization, and then serial dilution was performed, along with pH measurement. The plates were incubated at 35 ± 1ºC for 48 h. This test was carried out in triplicate.
-1 Variation in the averages of LAB growth in log CFU/mL was statistically assessed using the analysis of variance (ANOVA) of repeated measures, generalized linear models, and SPSS version 18 software (SPSS Inc., Somers, NY, USA). When a significant difference was found, the Bonferroni multiple comparison test was used, with a significance level of p ≤ 0.05.
We tested the tolerance of E. faecium M7AN7-1, E. faecium M7AN10, and L. rhamnosus Fagron® (a commercial probiotic bacteria used as a positive control) to refrigeration (6.6 ± 1.5 ºC) for 30 days. Weekly cell counts showed that the three LAB began -1 and completed tests with numbers higher than 8 Log CFU mL .
L. rhamnosus demonstrated the highest viability, followed by E.
faecium M7AN7-1 and then E. faecium M7AN10 (Table 1). Using the Bonferroni multiple comparison test, we observed a significant difference (p ≤ 0.05) between the variation in the averages of -1 L. rhamnosus growth in log CFU mL , and those of E. faecium M7AN10 and E. faecium M7AN7-1. This variation depended on the time when the viable cell count was done. However, since E.
faecium M7AN10 and E. faecium M7AN7-1 presented results -1 higher than 6 Log CFU mL (Table 1), they maintained sufficient viability to be considered stable cultures under refrigeration.
The cell viability of the E. faecium M7AN7-1, E. faecium M7AN10, and L. rhamnosus strains in frozen 20% powdered skim milk was tested over 90 days. The monthly viable cell counts demonstrated that, at the beginning of the experiment, all three LAB showed -1 cell viability higher than 8 Log CFU mL . These levels remained consistent after 90 days of being frozen (Table 2), with the E.
faecium M7AN7-1 strain presenting the highest numbers of viable cells, followed by L. rhamnosus and E. faecium M7AN10 respectively. Variation in the averages of L. rhamnosus growth in -1 log CFU mL was statistically different (p ≤ 0.05) from that of E.
faecium M7AN10 in all cell counts, except after 90 days of being frozen. Despite these variations, E. faecium M7AN10 maintained -1 cell viability and presented results greater than 6 Log CFU mL .
Monthly cell viability counts were performed on the freeze-dried E. faecium M7AN7-1, E. faecium M7AN10, and L. rhamnosus strains. The initial count was as follows: E. faecium M7AN7-1 9.14 Identical lowercase letters in the same column and identical uppercase letters in the same row indicate no statistical Identical lowercase letters in the same column and identical uppercase letters in the same row indicate no statistical
LAB Before refrigeration 7th day 8.53 ± E. faecium M7AN7-1 8.70 ± 0.04 abA 0.13 8.46 ± E. faecium M7AN10 8.48 ± 0.02 aA 0.03 8.60 ± L. rhamnosus 8.70 ± 0.01 bA 0.05 difference (p > 0.05) after using the Bonferroni multiple comparison test.
-1
LAB Before freezing 30th day aA E. faecium M7AN7-1 8.88 ± 0.07 8.54 ± 0.09 bA E. faecium M7AN10 8.41 ± 0.01 8.34 ± 0.01 aA L. rhamnosus 8.83 ± 0.04 8.53 ± 0.08 difference (p > 0.05) after using the Bonferroni multiple comparison test.
± 0.07, E. faecium M7AN10 8.50 ± 0.10, and L. rhamnosus 9.16 ± 0.01. Even though there was a small decrease after lyophilization, -1 the LAB maintained cell counts greater than 8 Log CFU mL (Table 3). L. rhamnosus showed the highest concentrations of viable cells 180 days after the lyophilization process, indicating the greatest tolerance to the process. This level of tolerance was followed by that of E. faecium M7AN7-1 and then E. faecium M7AN10. After using the Bonferroni multiple comparison test, we verified that the variation in the averages of L. rhamnosus growth -1 in log CFU mL differed significantly (p ≤ 0.05) from those of E. faecium M7AN10 in all test periods, always reaching more than 6 -1 Log CFU mL .
-1 In this test, the LAB presented values above 7 Log CFU mL when exposed to 0.1% bile salts. However, only the E. faecium M7AN7-1 and E. faecium M7AN10 strains remained viable after contact with (FAGRON) in refrigerated 10% powdered skim milk at 7, 14th day 21st day 30th day aB aAB abA aA 8.59 ± 0.02 8.42 ± 0.20 8.59 ± 0.01 bB bA aC aAB 8.44 ± 0.01 8.30 ± 0.07 8.46 ± 0.09 aA cA bA bB 8.72 ± 0.04 8.67 ± 0.09 8.81 ± 0.04
60th day 90th day aB aB aB 8.58 ± 0.01 8.50 ± 0.05 bB bAB bC 8.41 ± 0.05 8.16 ± 0.05 aB aB abB 8.57 ± 0.02 8.23 ± 0.27 0.3 and 0.5% bile salts for 4 h, demonstrating cell counts greater -1 than 6 Log CFU mL (Table 4). E. faecium M7AN10 remained stable after being exposed to 1% bile salts and reached cell counts -1 of 4 Log CFU mL , unlike L. rhamnosus that did not tolerate exposure to 0.3, 0.5, and 1% bile salts. In the control treatment (with no added bile salts), the cell counts were greater than 8 Log -1 CFU mL (Table 4). The Bonferroni multiple comparison test showed that the variation in the averages of L. rhamnosus growth -1 in log CFU mL differed significantly (p ≤ 0.05) from those of E. faecium M7AN7-1 and E. faecium M7AN10. This demonstrated that both LAB showed stability under the tested conditions.
The E. faecium M7AN10 and L. rhamnosus isolates maintained their viability throughout the test period, reaching cell counts -1 -1 above 7 Log CFU mL and 8 Log CFU mL , respectively.
10 10 However, E. faecium M7AN7-1 lost its viability after 90 minutes of Identical lowercase letters in the same column and identical uppercase letters in the same row indicate no statistical
LAB Freeze-drying (0 h) 30th day 60th day 8.65 ± 8.71 ± 8.72 ± E. faecium M7AN7-1 aA aE 0.02 0.02 0.05 8.49 ± 8.53 ± 8.47 ± E. faecium M7AN10 bA aBCDEF 0.08 0.10 0.09 9.01 ± 9.01 ± 9.06 ± L. rhamnosus aA bABE 0.20 0.11 0.01 difference (p > 0.05) after using the Bonferroni multiple comparison test.
The LAB remained viable after exposure and reached cell counts -1 above 8 Log CFU mL . The E. faecium M7AN7-1 isolate (FAGRON) in freeze-dried 20% powdered skim milk at 30, -1 .
90th day 120th day 150th day 180th day 8.89 ± 8.75 ± aE aF 8.86 ± 0.01 9.02 ± 0.02 aB aC aD 0.03 0.09 8.53 ± 8.43 ± bE bF 8.49 ± 0.01 8.45 ± 0.01 bB bC bD 0.01 0.01 9.11 ± 9.10 ± aE cF 8.94 ± 0.21 9.12 ± 0.05 cB cC cD 0.01 0.05 -1 of growth in log CFU mL were independent of the evaluation time, and we found a statistically significant difference (p ≤ 0.05) between the LAB.
Identical lowercase letters in the same column and identical uppercase letters in the same row indicate that no Identical lowercase letters in the same column indicate no statistical difference (p > 0.05) after using the Bonferroni multiple comparison test. For the control treatment: identical symbols in the same row indicate no statistical difference (p > 0.05).
-1.
60 min LAB 0 h Control SGF 7.18 ± aA+ a+ E. faecium M7AN7-1 7.96 ± 0.30 8.18 ± 0.05 aA 0.29 7.51 ± aA+ a+ E. faecium M7AN10 7.90 ± 0.20 7.20 ± 1.12 aB 0.18 8.15 ± 8.09 ± 8.18 ± L. rhamnosus aA+- a+- bA 0.04 0.03 0.09 statistical difference (p > 0.05) after using the Bonferroni multiple comparison test.
-1.
60 min 90 min LAB 0 h Control SIF Control Aa+ a- aB E. faecium M7AN7-1 8.31 ± 0.03 8.25 ± 0.09 8.40 ± 0.02 8.20 ± 0.07 bA+ b- bB E. faecium M7AN10 7.98 ± 0.13 7.97 ± 0.02 8.18 ± 0.01 8.03 ± 0.01 cA+ c- bA L. rhamnosus 8.13 ± 0.15 7.99 ± 0.05 8.22 ± 0.09 8.23 ± 0.02
All three LAB started the experiment with cell counts greater -1 -1 than 8 Log CFU mL . However, only 5 Log CFU mL of the L.
10 10 rhamnosus isolate were able to adhere to Vero cells during testing. The E. faecium M7AN7-1 and E. faecium M7AN10 strains showed more satisfactory adhesion to Vero cells, exhibiting a value of 6 -1 Log CFU mL . This demonstrated a superior adhesion capacity over that of L. rhamnosus (Figure 1). In terms of percentages of adherence, E. faecium M7AN7-1 presented 1.23% adhesion while E. faecium M7AN10 showed 0.33% adhesion. At the beginning of the experiment, the variation in the averages of L.
-1 rhamnosus growth in log CFU mL differed significantly (p ≤ 0.05) from that of E. faecium M7AN7-1. However, after 4 h of contact with Vero cells, the variation in the averages of growth of all the -1 LAB in log CFU mL was statistically different (p ≤ 0.05). Results showed that the E. faecium M7AN7-1 and E. faecium M7AN10 isolates presented viable cell counts that are suggestive of adhesive
90 min 180 min Control SGF Control SGF 3.32 ± a+ aB a+ 8.17 ± 0.02 5.82 ± 0.48 8.04 ± 0.16 aC 0.49 7.19 ± b- bAB a+- 6.08 ± 0.96 7.62 ± 0.35 6.87 ± 1.14 9b 0.5 B 8.12 ± a+ bA a8.20 ± 0.04 7.70 ± 0.83 8.15 ± 0.02 cA 0.03
180 min 240 min SIF Control SIF Control SIF a* aB a/ aC a! aC 8.44 ± 0.00 8.24 ± 0.04 8.58 ± 0.00 8.24 ± 0.04 8.60 ± 0.03 b* C b/ bD b! bD 8.42 ± 0.03ª 7.93 ± 0.11 8.32 ± 0.02 7.95 ± 0.06 8.28 ± 0.04 c* bA c/ cA c! bA 8.24 ± 0.04 8.11 ± 0.05 8.09 ± 0.09 8.14 ± 0.08 8.14 ± 0.13 -1 potential (i.e. equal to or greater than 6 Log CFU mL ).
The efficiency of microencapsulating Enterococcus faecium M7AN10 After the microencapsulation of the E. faecium M7AN10 and L. rhamnosus (control) isolates in 2% alginate, we tested the efficiency of the process. E. faecium M7AN10 presented 80.74% efficiency while L. rhamnosus showed 87.32% efficiency. Before undergoing microencapsulation, both LAB had cell counts above 8 Log CFU -1 mL . However, there was a reduction in the cell viability of both -1 after this procedure: L. rhamnosus reached 7 Log CFU mL and -1 E. faecium M7AN10 reached 6 Log CFU mL for E. faecium (Figure 2).
Microencapsulated LAB cultures in UHT whole milk and MRS broth were monitored. The release of bacterial cultures from the microcapsules occurred differently way for each of the LAB. The release of the L. rhamnosus bacteria occurred from the 15th day in UHT whole milk and the 7th day in MRS broth. At the -1 end of testing, only 5 Log CFU mL had been released in both media. In contrast, E. faecium M7AN10 bacteria were released from the microcapsules at zero time, both in the UHT whole milk and the MRS broth. One aspect that could explain this early result is the ability of viable E. faecium M7AN10 cells to adhere to the capsules. After 30 days, the viability of this strain was -1 -1 measured at 8 Log CFU mL in milk and 6 Log CFU mL in 10 10 MRS broth (Figure 3). The variation in the averages of the E.
-1 faecium M7AN10 growth in log CFU mL differed significantly (p ≤ 0.05) from that of L. rhamnosus, whether in relation to the UHT whole milk or the MRS broth and regardless of the moment of the viable cell count.
The 2% alginate microcapsules containing E. faecium M7AN10 -1 (6.51 ± 0.04 Log CFU mL ) were tested in a commercial UHT chocolate-flavored milk drink, and its release in this culture was assessed for 30 days. At the beginning of the test (time zero), 3.97 -1 ± 0.65 Log CFU mL of this bacterium had already been released in the UHT chocolate milk drink; however, on the 30th day, the viable cell count in this food matrix increased to 7.37 ± 0.45 Log -1 CFU mL (Figure 4). The pH was also measured during this experiment. At time zero the pH was 6, but from the 7th to the 30th day, it fell to 5.
The Enterococcus genus has often been isolated from samples of milk and dairy products [15-18]. Some research has aimed to assess and highlight the potential probiotic benefits of these microorganisms. As such, it is important to assess the stability of these isolates under conditions that mimic those of dairy product conservation and processing [19].
The ability of LAB to tolerate low temperatures is an essential question to address since the vast majority of products containing probiotic cultures are dairy derivatives that require storage under cold conditions [20-24]. The results of this study demonstrated that the LAB maintained cell viability after the experiments of refrigeration, freezing, and freeze-drying. These data corroborate those reported by other authors [7]. In their experiment, they assessed the ability of Bifidobacterium to maintain viability under refrigerated and frozen conditions. Viable cell counts -1 measured between 8.9 to 9.7 Log CFU mL after six months of testing at a temperature of -20ºC. The authors also reported viability after four weeks of testing at a temperature of 4 ºC, and viable cell counts ranged from 8.2 to 9.1 Log CFU mL . The lyophilization of LAB is an important process, as many industries market their goods in this way. Was evaluated the tolerance of encapsulated L.
M7AN10 and L. rhamnosus (control) before and after 4 h of contact
Figure 2: Viable cell count of L. rhamnosus and E. faecium M7AN10
Figure 3: Viable cell count of E. faecium M7AN10 and L. rhamnosus
casei DSPV 318T and L. plantarum DSPV 354T to lyophilization and its stability at a refrigerated storage temperature (4ºC). The authors found that the microorganisms maintained viability after lyophilization (regardless of the type of cryoprotectant) and after the freeze-dried capsules were placed in refrigerated storage for 84 days [25].
The cell viability of probiotic cultures should not only be studied within the context of food matrices. Transit through the gastrointestinal tract should also be considered. To offer benefit, probiotics must be able to tolerate the effects of bile salts, gastric fluid, and intestinal fluid [26]. For this reason, studies that evaluate probiotic potential should take into account the capacity of microorganisms to face these adverse conditions. E. durans LAB18s was isolated from Minas fresh cheese and tested it against different concentrations of bile salts (0.1, 0.25, 0.50, 1.0, and 1.5%) for 4 h. This bacterium was able to tolerate all concentrations during the experiment, and present counts greater -1 than 8 Log CFU mL [27]. Our results showed reduced viability since E. faecium M7AN7-1 and E. faecium M7AN10 registered -1 counts below 7 Log CFU mL after 4 h of contact with 0.5% bile salts. Although there was an increase in viability after exposure to 0.1 and 0.3% bile salts, only E. faecium M7AN10 registered -1 viable cell counts (4.42 ± 0.21 Log CFU mL ) after contact with 1% bile salts. E. faecium was isolated from artisanal cheeses and tested their tolerance to 0.3% bile salts. The authors found that 24 of 103 isolates were able to tolerate that concentration of bile salts for 240 min [28]. However, other authors demonstrated that E. faecalis UGRA10 lost its viability after exposure to 0.3% bile salts [29].
To evaluate tolerance to simulated gastric fluid (pH 2), were isolated four strains of E. faecium from human milk and found that after 1 h of contact with the simulated solution, cell counts -1 lowered from 8 Log CFU mL to approximately 2 to 3 Log CFU 10 10 -1 mL . After 3 h, counts were <1. Our results varied among the LAB. E. faecium M7AN7-1 presented a cell count of approximately -1 3 Log CFU mL , while E. faecium M7AN10 presented results -1 above 7 Log CFU mL after 3 h [30]. However, were published results that demonstrated the high viability of E. faecium SJRP20 and SJRP65. The authors performed a consecutive evaluation by simulating the gastric phase (pH 2.5), followed by a simulated enteric phase (pH 8).After the first phase, the viability of both strains -1 was greater than 9 Log CFU mL . After the second phase, the -1 cell count reached 8 Log CFU mL , approximately [31]. Similar results were obtained by other authors, that used a simulated gastric and intestinal transit test to assess L. paracasei. They reported cell count viability of approximately 8 Log CFU -1 -1 mL and 7 Log CFU mL for each of the phases [32].
However, some researchers have shown that tolerance to intestinal fluid is a more important property because some foods can act as buffers in the gastric tract and help probiotics reach the small intestine [27]. Was evaluated the viability of E. faecium exposed to -1 0.1 g mL of pancreatin at pH 8, for 240 min. They noted that 45% of the isolates showed tolerance to this adverse condition, showing survival rates ranging from 78.6 to 99.2%, depending on the strain [28]. The tolerance of L. plantarum BLS29 was assessed to intestinal fluid after 4 h of exposure and reported cell count viability above 8 -1 Log CFU mL . Maintaining viability after gastrointestinal transit is an important aspect because probiotic cultures need to colonize the intestine to fully exercise their functional potential [33].
The ability to adhere to epithelial cells is also an important property for a probiotic since this characteristic will determine its competence in colonizing the intestine. The results found in the literature are usually described as a percentage of adhesion. A research evaluated the adhesion capacity of L. plantarum LB95 and L. plantarum LB13 to HT-29 cells (human adenocarcinoma epithelial cells) and found that adhesion percentages ranged from 0.7 to 1.8%. In some situations, the ability to adhere may differ between certain types of epithelial cells, as well as between different LAB species belonging to the same genus [34]. The adhesion of Lactobacillus spp. to Vero and HeLa cells (human carcinoma epithelial cells) was evaluated. The authors observed that Vero cells adhered more efficiently than HeLa cells [12]. Still, on the topic of the different types of cells that have been used in research, was reported the adhesion of Leuconostoc mesenteroides subsp. mesenteroides, E. faecium, and six different L. plantarum isolates to Caco-2 cells (human colon adenocarcinoma epithelial cells). They considered high adhesion values to be between 4 and 8%, and their results showed adhesion levels ranging between 1.3 and 8% [13]. However, other work evaluated the adhesion of five E. faecium isolates to Caco-2 and TC7 cells (human colon adenocarcinoma epithelial cells). Three isolates presented adhesion percentages of 0.7, 0.9, and 1.4% (very low adhesion). One isolate showed 7% adhesion (moderate adhesion) while another demonstrated 21% adhesion (high adhesion) [35].
Regarding the adhesion percentages of our isolates, the E. faecium M7AN7-1 and E. faecium M7AN10 strains presented 1.23% and 0.33% adhesion, respectively. However, the results -1 of the viable cell count showed that 6.93 Log CFU mL of E.
-1 faecium M7AN7-1 and 6.14 Log CFU mL of E. faecium M7AN10 were able to adhere to Vero cells.
For a probiotic culture to maintain its viability after gastrointestinal transit, adhere to the intestine, and consequently colonize it, microencapsulation can be used to minimize the impacts caused by the adverse conditions of the gastrointestinal tract. In addition to protecting microorganisms from damage, microencapsulation helps probiotics maintain viability during the temperature fluctuations and pH changes that are typical of food production technologies and food storage [21,36].
Some encapsulation methods have demonstrated efficiency with some microorganisms, but not with others. E. faecium IS-27526 and L. plantarum IS-10506 probiotic strains were encapsulated in 4.75% alginate, using fluidized bed technology. They then tested these microcapsules in a system that simulated the gastric tract and small intestine. Their results showed that the L. plantarum IS- 10506 strain presented 84.5% viability, while only 15.7% of the E. faecium IS-27526 strain remained viable [37].
Some encapsulating materials can also enhance sustained LAB viability better than others.A research encapsulated L. acidophilus in separate alginate and carrageenan 2% concentrations, using an automated extrusion method. The carrageenan encapsulated the microorganism with 96% efficiency, while the alginate presented 98% efficiency [22].
Using encapsulated microorganisms in food is an option for extending viability beyond that of free-form probiotics. 3% alginate and the emulsion method were used to encapsulate L. salivarius CET 4063 and L. paracasei A13 before testing them in fermented milk. This dairy derivative was stored at 4 ºC, and the cell count viability of these microorganisms was evaluated for 56 days. The encapsulated LAB remained significantly (p ≤ 0.05) more viable than those in free form [21].
Ice cream has also been shown to be a good food matrix for encapsulated probiotics. To test increased viability, was used the emulsion process tocombine 3.6% alginate, 2% starch, and 6% glucose (a cryoprotectant) and encapsulate B. longum CFR815j. After 15 days of storage at -20 ºC, the results showed a 2.5% reduction in the viability of the encapsulated form of the probiotic. This was different from the 40% reduction in the viability of its free form [23]. Similarly, other research used separate 2% concentrations of alginate and carrageenan to encapsulate L. acidophilus, and study the microcapsules in ice cream. They monitored their viability for 120 days. The results showed that this bacterium remained significantly (p ≤ 0.05) more viable in its encapsulated form, whether coated in alginate or carrageenan, than in its free form. These data confirm that microencapsulation effectively maintains the viability of microorganisms in foods that need to be stored at low temperatures [22].
When we tested the encapsulated probiotic cultures in a UHT flavored milk drink and UHT whole milk, we noted that the microcapsules played an important role in maintaining the viability of E. faecium M7AN10. This efficiency was reflected in the cell counts because, during 30 days of storage in a refrigerator, -1 they were greater than 7 and 8 Log CFU mL , respectively. This established the necessary cell count viability for a probiotic bacterium. According to the Brazilian National Health Surveillance E. faecium is listed as a recognized probiotic. Our results showed that the E. faecium M7AN10 strain maintained the cell count viability attributed to probiotic cultures [38].
Based on the data of our in vitro tests, and comparisons with the information found in the literature, we suggest that the E. faecium M7AN10 strain may be a potential probiotic. However, in vivo tests must be carried out to see if it sustains probiotic characteristics and cell count viability.
This study explored the probiotic potential and stability of Enterococcus faecium isolates at different temperatures. Two LAB strains remained viable after maintenance and stability tests under different conditions. However, in tests for probiotic potential, E. faecium M7AN10 performed better after exposure to simulated gastric fluid.
E. faecium M7AN10 was microencapsulated in alginate, with good encapsulation efficiency. After this evaluation, E. faecium M7AN10 was tested in UHT milk. After 30 days in this food, the cell count viability of this bacterium was measured at values above 7 -1 Log CFU mL .
The results we obtained after identifying isolates of native lactic acid bacteria from this underexplored raw material highlight the importance of searching for new isolates that may show promising probiotic potential. Giving visibility to the natural functional potential of buffalo milk will promote interest in the development of naturally functional buffalo dairy products. Further research is needed to study these isolates in vivo.
We thank the Virology Laboratory of the Veterinary Faculty of the Federal University of Rio Grande do Sul.
The authors declare that there is no conflict of interest